title: Comprehensive analysis of neurotransmitters from regenerating planarian extract using an ultrahigh-performance liquid chromatography/mass spectrometry/selected reaction monitoring method.
abstract: BACKGROUND
  Absolute quantification of neurotransmitters (NTs) from biological systems is imperative to track how changes in concentration of active neurochemicals may affect biological behavior. A sensitive method for the absolute quantification of multiple NTs in a single method is highly needed.
  
  
  METHODS
  A stable-isotope dilution ultrahigh-performance liquid chromatography/mass spectrometry/selected reaction monitoring (UHPLC/MS/SRM) assay has been developed for a sensitive and quantitative assessment of NTs in planaria. We used this method for the simultaneous quantification of 16 NTs. All analytes showed a linear relationship between concentrations (0.78-50 ng/mL), regression coefficients higher than 0.97, accuracy (91-109%) and low coefficients of variation (CVs). The inter-day CVs for the lowest quality controls (1.56 ng/mL) were in the range between 2-11%.
  
  
  RESULTS
  The levels of most of the NTs were similar in both sexual and asexual planarians except for glutamic acid, which was about two-fold higher in asexual compared to sexual planarians. We identified high levels of serotonin and failed to detect tryptamine suggesting that the pathway essential for the conversion of tryptophan into tryptamine is absent in planarians. Interestingly, we also found high levels of dopamine and L-DOPA in regenerating planarians suggesting their possible role in regeneration.
  
  
  CONCLUSIONS
  For the first time, we developed novel methodology based on UHPLC/MS/SRM and quantified 16 NTs with high sensitivity and specificity from sexual and asexual strains of planarian Schmidtea mediterranea. This method will also have great application in quantifying various NTs with great precision in different model systems.
authors.affiliation: Centre for Cellular and Molecular Platforms, National Centre for Biological Sciences, GKVK, Bellary Road, Bangalore, 560065, India.
authors.forename: Kannan
authors.initials: K
authors.lastname: Rangiah
authors.forename: Dasaradhi
authors.initials: D
authors.lastname: Palakodeti
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D018698:Glutamic Acid / Q000032:analysis / Q000302:isolation & purification; D057230:Limit of Detection; D018377:Neurotransmitter Agents / Q000032:analysis* / Q000302:isolation & purification; D010932:Planarians / Q000737:chemistry* / Q000502:physiology; D012098:Reproduction; D012100:Reproduction, Asexual; D012701:Serotonin / Q000032:analysis / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014363:Tryptamines / Q000032:analysis / Q000302:isolation & purification
chemical_list: D018377:Neurotransmitter Agents; D014363:Tryptamines; D012701:Serotonin; D018698:Glutamic Acid; C030820:tryptamine